PROJECT SUMMARY/ABSTRACT Lipid-enriched extracellular lamellar membranes in the outermost layer of skin, i.e., stratum corneum (SC), subserve epidermal permeability barrier function, as required for mammalian survival in a dry environment. A family of 10 ceramides (Cer) dominates in these membranes, accounting for about H50% of SC lipid, and therefore H5% of the total weight of SC. Not only their quantities, but also their molecular heterogeneity is required to form lamellar membrane structures. In particular, w-O-acylCer (or acylCer), that contains an acyl group esterified to the w-hydroxy terminal of amide-linked very long-chain fatty acids (>C28), is not only unique to the epidermis, but also critical for normal permeability barrier homeostasis. Importantly, a selective deficiency in acylCer occurs in atopic dermatitis (AD), which could further aggravate inherent defects in SC structures. We showed that inhibition of w-hydroxylation decreases acylCer production, provoking barrier abnormalities, and that mice lacking the normal FA elongase, elongation of very long chain FA (or ELOVL) 4, display a lethal post-natal barrier defect; neither acylCer nor the w-OH Cer covalently-attached to cornified envelope proteins (corneocyte lipid envelope, CLE) are formed in these mice. These acylCer are essential for both extracellular lamellar membrane organization, and for corneocyte lipid envelope formation. We recently showed that both acyl-CoA wax alcohol acyltransferases (AWAT) 1 and CGI-58 (a cofactor of triacylglycerol lipase) are further required for w-O-esterification, leading to acylCer production. Yet, neither the basis for acylCer deficiency in AD, nor the contribution of acylCer deficiency to the pathogenesis of AD is known. We hypothesize that either decreased synthesis or accelerated hydrolysis of acylCer occurs in AD. This biochemical abnormality can be attributed to increased T helper cell 2 (Th2) cytokine-induced downregulation and/or aberrant xeric stress-mediated-signaling of acylCer production via the external humidity and osmotic sensors, TRPV4 and TonEBP, respectively. Together, these signaling defects account for deficiency of acylCer in AD. We will investigate 1) the enzymatic (key enzymes/cofactor, i.e., ELOVL4, w-hydroxylase, AWAT1, and CGI-58) basis for acylCer deficiency as well as the structural/functional consequences of acylCer deficiency in AD; 2) how Th2 cytokines downregulate acylCer in AD; 3) how xeric stress regulates acylCer synthesis via the TRPV4 receptor and/or TonEBP signaling and their alterations in AD; and 4) novel therapeutic strategies for acylCer restoration in AD.